Protein synthesis in bluetongue virus-infected cells.

The first virus-specific proteins in bluetongue virus (BTV)-infected cells can be demonstrated 2 to 4 hr after infection (p.i.) at 31° by immune precipitation. There is a rapid increase in the rate of synthesis until 11 hr p.i. after which synthesis remains at about the same level until 26 hr p.i. Apart from the seven BTV capsid polypeptides, two noncapsid polypeptides P5A and P6A, with respective molecular weights of 54,000 and 40,000 daltons, were identified in the infected cells. Polypeptide P5A is synthesized at a higher rate than any of the others. It is the main if not only component of a complex with an S value slightly less than 400. There is little evidence for specific regulation of the translation process in BTV-infected cells. With the possible exception of polypeptide P1, all the others are translated with a relative frequency that is not significantly different from that of the corresponding mRNA species. Pulse-chase experiments indicate that the BTV polypeptides do not accumulate in the soluble fraction in the same ratio in which they are synthesized. Polypeptide P3 is removed from the soluble protein pool much more rapidly than any of the other capsid polypeptides. These pulse-chase experiments provided no evidence for a precursor-product relationship between any of the BTV polypeptides. The possibility that one of the noncapsid polypeptides, P6A, is a modified form of one of the capsid polypeptides can, however, not be excluded.     

Protein synthesis in Sendai virus-infected cells.

The rate of protein synthesis in chicken embryo cells infected with Sendai virus 18 to 20 h previously was about two times greater than in mock-infected controls. At this time of infection six stable virus-induced proteins, four major structural proteins (P, NH, NP and M) and two non-structural proteins (28K and 61K), were identified by electrophoresis in SDS-polyacrylamide gel of total cell extracts. The structural glycopeptide F was not detected in the infected cell extracts. Pulse-chase experiments showed that PN NP, M and 28K proteins either did not undergo any post-translational processing or the processing occurred very rapidly. By contrast, a glycopeptide NH was apparently derived from one of two unstable precursons, 69K or 63K, which were revealed only after a short pulse. The synthesis of virus-specific proteins appeared to be regulated since its rate varied for individual classes of proteins. In nucleocapsid-particles isolated from infected cells two major structural proteins (P and NP) were found. A minor component with a very large mol. wt. was revealed in these particles as well as in the virus particle.     

Protein synthesis in vaccinia virus-infected cells

Summary Human Hep-2 cells were submitted to hypertonic shock (210 mM NaCl) to block host protein synthesis before infection with vaccinia virus. With the start of infection, the medium isotonicity (116 mM NaCl) was restored, and the effect of viral infection on the recovery of host polyribosomes and protein synthesis was studied. Although host translation blockage was released together with infection, vaccinia virus did not affect immediately host protein synthesis. During the first hour of recovery, infected cells could perfectly rebuild the polyribosome profile and recuperate the rate of protein synthesis. Also, during recovery, formation of the initiation complex for protein synthesis was not affected by viral infection. In this period, viral mRNA and proteins were detected by slot blot and SDS-polyacrylamide gel electrophoresis. The inhibitory effect of vaccinia virus on host translation was observed after the second hour of infection. These findings suggest that vaccinia virus-mediated shutoff occurs in a later period during infection, in parallel with viral mRNA accumulation in the polyribosomes and after the on-set of viral DNA replication.     

Protein synthesis in Japanese encephalitis virus-infected cells

We studied the effects of actinomycin D, cycloheximide, and puromycin on virus-specified protein synthesis in Japanese encephalitis (JE) virus-infected chick embryo and LLC-MK₂ (rhesus monkey kidney) cells. To prove that the radioactively labeled proteins we had previously identified in infected chick cells were virus-specified, we showed that they electrophoretically comigrated with radioactively labeled proteins from infected, but not from uninfected, LLC-MK₂ cells. We found that the maximum value for the ratio of protein synthesis in infected chick cells to uninfected cells occurred when the cells were treated with actinomycin D and were pulse-inhibited with cycloheximide. Alone, actinomycin D treatment decreased the background radioactivity of high molecular weight in electropherograms of infected chick cells and allowed virus-specified proteins to be prominent. Cycloheximide pulse-inhibition of infected, actinomycin D-treated cells decreased total cellular protein synthesis and slightly decreased the background radioactivity in electropherograms without changing the distribution of radioactivity among virus-specified proteins. Neither drug treatment decreased the yield of infectious virus. These results differ in some respects from the related results of Trent and Qureshi (1971). In contrast to our results with cycloheximide, pulse-inhibition of infected chick embryo cells with puromycin inhibited the synthesis of polypeptides NV-5, NV-4, and NV-1 (virus-specified nonstructural, nonglycosylated proteins) to a greater extent than that of V-3, NV-3, NV-2, and V-2 (virus-specified glycosylated and/or structural proteins). It also generally inhibited the synthesis of large proteins relative to small ones.We then studied the effects of puromycin and cycloheximide in LLC-MK₂ cells. In contrast to our results in chick embryo cells, pulse-inhibition of infected LLC-MK₂ cells with either drug (in the continuous presence of actinomycin D) did not alter the pattern of virus-specified proteins in electropherograms from that obtained without pulse-inhibition. Treatment with continuous levels of either drug (in the presence of actinomycin D) did, however, alter the protein pattern by differentially inhibiting the synthesis of nonstructural, nonglycosylated proteins. By labeling infected cells with one of eight different amino acids, we were unable to find an unusually enriched (or lowered) amino acid content that was common to all nonstructural, nonglycosylated proteins. A possible explanation for the differential inhibition is that the virus directs the formation of functionally different polyribosomes, messengers, or initiation factors which vary in their susceptibility to low levels of inhibitors of protein synthesis.     

Protein synthesis in Rift Valley fever virus-infected cells

The complete sequence of the neuraminidase (NA) gene of the influenza A strain A/parrot/ Ulster /73 ( H7N1 ) has been determined after reverse transcribing and cloning it into the pBR322 plasmid, followed by subcloning into M13 vectors and sequencing with dideoxynucleotide chain terminators. The gene consists of 1458 nucleotides and codes for a protein of 469 amino acids. The neuraminidase has seven potential glycosylation sites. According to the molecular weight as determined by electrophoretic migration in polyacrylamide gel all of these sites might carry a carbohydrate side chain. When the parrot Ulster NA was compared with two other N1 neuraminidases, those of the human PR8 and WSN strains, deletions in the stalk region of 15 amino acids for PR8 NA and of 16 amino acids for WSN NA were apparent. No further rearrangements were found within N1 neuraminidases. Although the parrot Ulster strain was isolated 40 years after the two human strains, the base sequence homology of their NA genes is still 83 or 82%, respectively.     

Protein synthesis in cucumber mosaic virus-infected tobacco leaves,pre-infected cells and protoplasts

Summary Polypeptide synthesis has been investigated in cucumber mosaic virus-infected tobacco by radiolabelling and polyacrylamide gel electrophoresis. In addition to the coat protein polypeptide of molecular weight 27,000, others of higher molecular weightca. 120,000 to 160,000 (probably two) and possibly one ofca. 32,000–34,000 were detected in the particulate fractions only. A polypeptide ofca. 53,000–57,000 was particularly evident in leaves systemically infected using a differential temperature treatment. Two polypeptides of low molecular weight,ca. 23,000–24,000 and 21,000, were only observed in pre-infected cells and protoplasts and probably represent coat protein degradation products.With 4 Figures     

Viral RNA synthesis in measles virus-infected cells

Sedimentation analysis of measles virus-specific cytoplasmic RNA at different times after infection revealed several size classes. The three specific components found in greatest amount were estimated to have sedimentation coefficients of 20 S, 27 S, and 35 S. A fourth component, 52 S, sedimented with characteristics of viral RNA. 20 S RNA appeared later than the other size classes, and accounted for an increasingly greater proportion of the total. However, with a temperature-sensitive mutant synthesis of all three major components was turned off simultaneously by transfer of infected cultures from permissive to nonpermissive temperature. Subgenomic-size RNA was found in virion preparations made by undiluted passage, but not after serial passage of diluted inoculum.     

Polypeptide synthesis i mumps virus-infected cells

The synthesis of polypeptides in infected Vero cells has been studied with two tissue culture adapted strains of mumps virus. Minor differences between the strains are found in the mobilities of the virus-specific polypeptides in SDS-PAGE. Eight virus-induced polypeptides have been identified with mol. wt. of 180K, 80K, 71K, 69K, 45 K, 39K, 23K and 17K. These may correspond to the L, HN, N, Fo, P, M, C and S polypeptides identified in other paramyxoviruses. The 69K F0 glycoprotein is probably a precursor for glycopolypeptides F1 and F2 with mol. wt. 61K and 14K, respectively.     

Deoxyribonucleic acid synthesis in vaccinia virus-infected HeLa cells

The control of DNA synthesis by the addition of graded concentrations of thymidine to HeLa cell cultures inhibited by 5-fluorodeoxyuridine has been determined to be a regulation at the cellular level and not a population selection process. In infected cells the rate of of vaccinia virus DNA in the presence of FUDR is not a function of the thymidine concentration. Instead, suboptimal thymidine levels were utilized preferentially to support optimal viral DNA synthetic rates which ceased abruptly when depletion of thymidine occurred. When FUDR was added early in the infectious cycle, the reduced yield of infectious virus obtained was also formed at a maximal rate.     

Enhancement of polysome formation and protein synthesis in Sendai virus-infected cells.

A considerable stimulation of total protein synthesis in Sendai virus-infected chicken fibroblasts occurred 18 h after infection. The stimulation of synthesis was shown to be due mainly to the synthesis of virus-specific proteins; the synthesis of host proteins was either unchanged or slightly enhanced. The level of functional polyribosomes in virus-infected cells was also increased as compared to that in mock-infected control while the number of free 80S ribosomes was proportionally decreased.     

Protein synthesis in virus-infected plants. 3. Effects of tobacco mosaic virus mutants on protein synthesis in Nicotiana tabacum

Nicotiana tabacum, infected with three widely differing chemically produced and one natural mutant of tobacco mosaic virus (TMV), was pulse labeled with [³H]leucine. Uninfected plants were similarly pulse labeled with [¹⁴C]leucine. The ³H- and ¹⁴C-labeled leaf tissues were combined, fractionated into subcellular fractions, denatured, dialyzed, and electrophoresed on polyacrylamide gels in SDS. Increases or decreases in the ${}^3\text{H}{}^{14}\text{C}$ ratio in gel slices indicated changes in the proteins synthesized in the infected fractions as compared to the uninfected control fractions.In contrast to wild-type TMV, where only coat protein was detected (Singer, 1971), protein synthesis differed markedly among the mutants and their subcellular fractions in both the number and molecular weights of the protein peaks. No protein was found to be common to all strains in the same subcellular fractions except the coat protein. In addition, decreases in the ${}^3\text{H}{}^{14}\text{C}$ ratio were found, indicative of suppression of specific host proteins. The total molecular weights of proteins appearing upon infection with some mutants exceeded the coding capacity of the viral nucleic acid, and there was no indication that any of the very large proteins found were precursors. It appears probable that these multiple responses specifically produced by mutants are the result of changes in host protein synthesis caused by the infection.     

The synthesis of polypeptides in influenza C virus-infected cells

The synthesis of virus-specific polypeptides was analyzed in MDCK cells infected with the JJ/50 strain of influenza C virus. In addition to three major structural proteins gp88, NP, and M, the synthesis of five polypeptides with molecular weights of 29,500 (C1), 27,500 (C2), 24,000 (C3), 19,000 (C4), and 14,000 (C5) was found in infected cells. None of these polypeptides were detected either in virions or in immunoprecipitates obtained after treatment of infected cell lysates with antiviral serum, suggesting that they are not viral structural proteins. Polypeptides C1-C5 were found to be synthesized in MDCK cells infected with different influenza C virus strains as well as in different host cell types infected with C/JJ/50. Further, it was observed that cellular protein synthesis was greatly reduced under hypertonic conditions, whereas the synthesis of C1-C5 was relatively unaffected. These results suggest that polypeptides C1-C5 are virus coded rather than host cell coded. Peptide mapping studies showed that each of polypeptides C3, C4, and C5 had a peptide composition similar to the M protein. The amount of C2 synthesized in infected cells was insufficient for mapping. This polypeptide was, however, found to rapidly disappear in pulse-chase experiments, suggesting that C2 is probably not unique but biosynthetically related to one of the other proteins. In contrast to these polypeptides, polypeptide C1 showed a map which is largely different from any major structural polypeptide. It therefore appears likely that C1 is a nonstructural protein of influenza C virus similar to the NS1 protein of influenza A and B viruses.